RESEARCH ARTICLE Copy number variation in the susceptibility to systemic lupus erythematosus Fernanda Bueno Barbosa1, Milena Simioni2, ClaÂudia EmõÂlia Vieira Wiezel1, FaÂbio Rossi Torres2, Miriam Coelho Molck2, Melvin M. Bonilla3, TaÃnia Kawasaki de Araujo2, 4 2 3 Eduardo Antoà nio Donadi , Vera Lu cia Gil-da-Silva-LopesID , Bernardo Lemos , Aguinaldo 1 Luiz SimõesID * 1 Department of Genetics, Ribeirão Preto Medical School, USP, Ribeirão Preto, SP, Brazil, 2 Department of Medical Genetics, Faculty of Medical Sciences, UNICAMP, Campinas, SP, Brazil, 3 Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, United States of America, a1111111111 4 Division of Clinical Immunology, Department of Medicine, Ribeirão Preto Medical School, USP, Ribeirão a1111111111 Preto, SP, Brazil a1111111111 a1111111111 * [email protected] a1111111111 Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic compo- OPEN ACCESS nent and etiology characterized by chronic inflammation and autoantibody production. The pur- Citation: Barbosa FB, Simioni M, Wiezel CEV, pose of this study was to ascertain copy number variation (CNV) in SLE using a case-control Torres FR, Molck MC, Bonilla MM, et al. (2018) design in an admixed Brazilian population. The whole-genome detection of CNV was per- Copy number variation in the susceptibility to systemic lupus erythematosus. PLoS ONE 13(11): formed using Cytoscan HD array in SLE patients and healthy controls. The best CNV candi- e0206683. https://doi.org/10.1371/journal. dates were then evaluated by quantitative real-time PCR in a larger cohort or validated using pone.0206683 droplet digital PCR. Logistic regression models adjusted for sex and ancestry covariates was Editor: Xu-jie Zhou, Peking University First applied to evaluate the association between CNV with SLE susceptibility. The data showed a Hospital, CHINA synergistic effect between the FCGR3B and ADAM3A loci with the presence of deletions in Received: April 4, 2018 both loci significantly increasing the risk to SLE (5.9-fold) compared to the deletion in the single Accepted: May 30, 2018 FCGR3B locus (3.6-fold). In addition, duplications in these genes were indeed more frequent in healthy subjects, suggesting that high FCGR3B/ADAM3A gene copy numbers are protec- Published: November 28, 2018 tive factors against to disease development. Overall, 21 rare CNVs were identified in SLE Copyright: © 2018 Barbosa et al. This is an open patients using a four-step pipeline created for identification of rare variants. Furthermore, het- access article distributed under the terms of the Creative Commons Attribution License, which erozygous deletions overlapping the CFHR4, CFHR5 and HLA-DPB2 genes were described permits unrestricted use, distribution, and for the first time in SLE patients. Here we present the first genome-wide CNV study of SLE reproduction in any medium, provided the original patients in a tri-hybrid population. The results show that novel susceptibility loci to SLE can be author and source are credited. found once the distribution of structural variants is analyzed throughout the whole genome. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Microarray data are available from BIPMed (http://bipmed.iqm.unicamp.br/lupus/). Funding: This work was supported by grants from FundacËão de Amparo à Pesquisa do Estado de São Introduction Paulo (FAPESP, grant number 2011/23794-7; Systemic lupus erythematosus (SLE, MIM 152700) is an autoimmune polygenic disease char- http://www.fapesp.br/), CoordenacËão de AperfeicËoamento de Pessoal de NõÂvel Superior acterized by local or systemic inflammation from the production of autoantibodies and (http://www.capes.gov.br/), FundacËão de Apoio ao immune complex deposition in several tissues [1]. SLE has a wide range of clinical manifesta- Ensino, Pesquisa e Assistência do HCFMRP-USP tions such as malar rash, discoid lesions, nephritis, and arthritis [2]. SLE is more prevalent in PLOS ONE | https://doi.org/10.1371/journal.pone.0206683 November 28, 2018 1 / 14 CNV profile in SLE (http://www.faepa.br/), and Conselho Nacional de women than in men at a rate of 9:1, and onset predominantly during childbearing age [3]. In Desenvolvimento CientõÂfico e TecnoloÂgico (CNPq; general, it is less prevalent in European ancestry populations than in African-Americans, Afri- http://cnpq.br/). FBB (grant numbers 2013/17062- can-Caribbean, Asians, and Hispanics [4]. 9, 2016/10306-8) is currently supported by a research fellowship from FAPESP, while ALS (grant In addition to the genetic component; hormonal, environmental, epigenetic and immuno- number 312547/2009-9), EAD (grant number logical factors contribute to the complex etiology of SLE [5]. High heritability and increased 304931/2014-4) and VLGSL (grant number concordance rates were identified in monozygotic twins (24±57%) compared with dizygotic 304455/2012-1) are supported by CNPq/Brazil. twins or full siblings (2±5%), suggesting that SLE has a complex genetic basis with sizable Competing interests: The authors have declared genetic and environmental components [6, 7]. Linkage and genome-wide association studies that no competing interests exist. conducted in cohorts of patients with SLE have confirmed that HLA and other loci are associ- ated with SLE [8]. This method has also been useful in identifying new candidate single nucle- otide polymorphisms (SNPs) correlated with the disease [9, 10]. In addition to SNPs, genomic segments that vary in copy number in relation to a reference genome (denoted as copy number variations, or CNVs, and typically greater than 50 bp [11]) have been associated with susceptibility to autoimmune diseases, including SLE [2]. Well-doc- umented CNVs that increase risk for SLE include deletions in C4 [12] and FCGR3B [13] genes, while CNVs in other genes were associated with SLE in single-population studied, e.g., TLR7 [14], DEFB4 [15], RABGAP1L [16] and HLA-DRB5 [17]. The small number of large-scale studies relating CNVs to SLE remains a significant gap in the genetic analysis of the disease [16, 18]. Additionally, the ancestral composition of popula- tions can often modify the results of association tests, such that the study of admixed popula- tions can produce different or even conflicting results compared with those reported in the literature [19, 20]. Based on the hypothesis that new SLE-related loci remain to be discovered using CNV approach, this study has evaluated the role of structural variation in SLE through genome- wide screening in Brazilian SLE patients. Materials and methods DNA samples The total case group comprised 135 unrelated SLE patients treated at the Collagen Disease Outpatient Clinic of the University Hospital at the Ribeirão Preto Medical School (HCFMRP, USP) in Brazil. All patients fulfilled the American College of Rheumatology revised criteria for SLE diagnosis [21]. The healthy control (HC) cohort includes 200 healthy unrelated subjects resident in São Paulo state, Brazil. DNA was extracted from the blood samples of SLE patients and control subjects using salting-out method [22] and QIAamp DNA Blood Maxi Kit (QIA- GEN, Hilden, Germany), respectively. For the array analysis, we selected a subgroup of 23 lupus nephritis patients (18 female, mean age at diagnosis: 31 ± 13 years) from the total of 135 SLE patients (125 female, mean age at diag- nosis: 32 ± 12 years). The frequency of clinical characteristics for the group and subgroup of SLE patients was, respectively: (1) nephritis: 61%/100%, (2) arthritis: 61%/74%, (3) malar rash: 38%/48%, (4) oral ulcers: 13%/13%, (5) photosensitivity: 40%/52%, (6) convulsions and/or psy- choses: 21%/30%, (7) hematologic disorder (hemolytic anemia or leukopenia < 4.000/mm3, lymphopenia < 1.500/mm3, and/or thrombocytopenia < 150.000/mm3): 57%/52%, (8) immu- nologic disorder (anti-dsDNA, anti-Sm, and/or anti-phospholipid): 73%/78%. Ethics The study design was approved by the Research Ethics Committee of FMRP/USP (CAAE: 03199712.0.0000.5440) and FCM/UNICAMP (CAAE: 03199712.0.3001.5404). All subjects enrolled in this research have signed the consent form approved by the ethics committees. PLOS ONE | https://doi.org/10.1371/journal.pone.0206683 November 28, 2018 2 / 14 CNV profile in SLE Cytoscan HD array The genome-wide human Cytoscan HD array (Affymetrix, CA, USA) was used to detect CNVs in SLE patients (n = 23) and healthy controls (n = 110) according to the manufacturer's proto- col. Scanned data files were generated using Affymetrix GeneChip Command Console software v. 1.2 and analyzed by Affymetrix Chromosome Analysis Suite (ChAS) software v. 3.0. CNV detection To calculate copy number regions throughout the genome the data were normalized to base- line intensities according to an internal reference model of ChAS software that comprised 270 HapMap samples and 96 other healthy subjects from BioServe Biotechnologies (BioServe, Beltsville, USA). CNVs regions were mapped according to the human reference sequence ver- sion GRCh37/hg19. The number of consecutive probes required defining each deletion or duplication and was limited to a minimum of 25/ 50 consecutive probes, respectively. After variant detection by ChAS, CNV distribution per subjects and chromosomes was analyzed using Plink v.1.07 [23]. Determination of CNV regions. Plink was used to evaluate the recurrence of common CNVs, CNV regions (CNVRs), which are the union of overlapping CNVs among subjects [24].